抗肉毒毒素A人源单链抗体在酵母细胞中的分泌表达  

High-level Secretion Expression of Human ScFv Against Botulinum Neurotoxin A in Pichia pastoris*

作　　者：王慧[1] 荫俊[1] 侯晓军[1] 邢洪光[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071

出　　处：《微生物学通报》2005年第2期50-53,共4页Microbiology China

基　　金：国家重点基础研究973项目资助(No.2002CB513205).

摘　　要：将特异肉毒抗毒素基因克隆入载体pPIC9k,G418抗性加压筛选阳性整合克隆,在毕赤酵母细胞GS115中进行分泌表达。获得了稳定分泌表达ScFv的工程菌,SDS-PAGE分析可见,目的蛋白分子量约为26kD,通过放大体积来探索重组抗毒素的诱导表达条件及纯化工艺,结果发现,1%甲醇诱导后72～84h,目的蛋白的表达达到高峰,占酵母培养上清中总蛋白的15%以上,经两步层析纯化,目的蛋白纯度可达95%。竞争活性测定结果表明,重组抗毒素在体外具有良好的活性,可竞争肉毒抗毒素马血清与毒素的特异结合。The specific ScFv gene against botulinum neurotoxin A (BoNTa)was cloned into pPIC9k. Positive integrators were screened by increasing the dose of G418 in culture and expressed in Pichia pastoris GS115. As a result, engineered recombinant clone were obtained. 26 kD product of interest was seen easily in SDS-PAGE. Expression of human ScFv got the highest level 15% of total secreted proteins during 72～84 h after 1% methanol inducing. Purification of ScFv was finished by two steps: gel filter and ion exchange. Competing ELISA showed that recombinant ScFv could compete with antiserum to specific bind BoNTa.

关 键 词：人源单链抗体 分泌表达 纯化 毕赤酵母 

分 类 号：R392[医药卫生—免疫学]